Two general procedures are currently available for the frozen preservation of living cell suspension. One requires the addition of high concentration of low molecular weight solutes that penetrate the cells. Although storage can be conducted in mechanical freezers, these storage procedures require specialized equipment and techniques to remove the solutes following thawing. The second utilizes high molecular weight polymers which do not enter the cells plus a lower concentration of penetrating solutes. Post-thaw processing in this case is much simpler but storage must be at very low temperature, usually in liquid nitrogen.
The formation of ice, which is a prime concern in any method of cell cryopreservation, is initiated by ice crystal nuclei. These may be foreign particles, regions of the surface of the container or dissolved molecules that have on their surface an array of hydrophilic residues comparable to the crystal structure of ice and thereby provide a template for the growth of ice. As a solution is supercooled below its nominal freezing temperature, the size of the array necessary to nucleate ice becomes smaller and, for pure water, the size of a critical nucleus approximates random aggregations of water and self-nucleation occurs. The temperature of self-nucleation is generally referred to as the homogeneous nucleation temperature (T.sub.hom). T.sub.hom for pure water is -40.degree. C. Just as the melting point of a solution is lowered by the addition of solutes, T.sub.hom is also lowered as the solute concentration increases.
As an aqueous solution is cooled, it becomes increasingly viscous until, at some low temperature, the translational movement of water molecules stops and the solution becomes a glass. This glass transition temperature, T.sub.g, rises as the solute concentration is increased. When freezing occurs during cooling, the solute concentration increases as the temperature falls. Experimentally, the equilibrium glass transition temperature is the temperature at which an aqueous solution, in equilibrium with ice, undergoes a second order phase transition from liquid to an amorphous solid. T.sub.g for different solutes are readily measurable, for example, by differential scanning calorimetry (DSC), and can vary from below -100.degree. C. for low molecular weight solutes to above room temperature for some complex polymers.
When a cell suspension is frozen, heterogeneous nuclei in the extracellular solution initiate ice. It is believed that living cells do not contain heterogeneous nuclei. As the ice crystals grow, water is removed from the extracellular solution, thereby increasing its osmolality. This in turn leads to a movement of water from within the cell down the osmotic gradient. As the temperature falls and ice grows, the cell is progressively dehydrated and at some point cell injury results. The nature of the dehydration injury is believed to be the result of membrane stresses leading to membrane rupture. This form of injury can be prevented by the addition of solutes at a multi-molar concentration so that the amount of ice formed is insufficient to result in damaging cell dehydration. Such a solute, to be useful, must be non-toxic at high concentration and must freely penetrate the cell. Both glycerol and dimethylsulfoxide (DMSO) have been used for this purpose. Glycerol is remarkably non-toxic at high concentrations but penetrates cell membranes slowly and is therefore difficult to introduce and remove. Dimethylsulfoxide penetrates rapidly but becomes increasingly toxic as concentrations exceed 1M (about 7%).
The necessary concentration of cryoprotectant can be substantially reduced by accelerating the rate of freezing. Since the movement of water from the cell interior, across the cell membrane and through the intervening solution to the ice crystal is a physical process requiring time, cell dehydration can be minimized by cooling at a rate that provides insufficient time for all freezable water to leave the cell.
This approach contains practical limitations, however, in that the intracellular solution remains dilute and it is more likely that intracellular ice will form. The goal of this method of cryopreservation is to find a cooling rate such that dehydration is insufficient to cause injury but still concentrates the intracellular solution enough to forestall intracellular freezing. For most cells, these two forms of injury overlap and there is no intervening window that avoids injury. It is then necessary to add penetrating cryoprotectants that reduce the amount of extracellular ice formed and thereby reduce cell dehydration, while at the same time increasing the intracellular concentration to make intracellular crystallization less likely. Concentrations of DMSO ranging from 5% to 10% have provided sufficient recovery of platelets and of stem cells to be clinically useful. However, it is undesirable to transfuse the quantities of DMSO involved and removing the DMSO prior to transfusion is inconvenient and results in the loss of cells. Takahashi (Japanese Journal of Freezing and Drying (1989) 35: 32-38) has reported the ability to reduce the amount of DMSO required to 2% when freezing monocytes in the presence of a 20% solution of extracellular polymeric cryoprotectant.
For many years it has been known that certain polymers are cryoprotective. These have included polyvinylpyrrolidone (PVP), dextran, and more recently, hydroxyethyl starch (HES). Since these large molecular weight polymers do not enter the cell, the mechanism by which they confer cryoprotection has been the subject of speculation.
Success of cryopreservation with certain water soluble polymers which do not permeate the cells has been reported for preservation of bacteria, erythrocytes, lymphocytes, platelets, bone marrow cells, fibroblasts, and other cells. Numerous theories have been advanced to explain this phenomenon including that the polymers protect cells by lowering extracellular salt concentrations at subfreezing temperatures just as penetrating cryoprotectants do, or, that the polymers might adsorb to cells and thus protect the membrane in some way. It has also been speculated that during freezing an electrolyte gradient develops from inside to outside the cells causing an electrolyte leakage which relieves osmotic stress. Phagocytosis of the polymers has even been suggested, which would have the effect of converting them into intracellular agents.
None of these hypotheses explains, however, why some polymers are cryoprotectants and others are not. Nor do they explain why low molecular weight non-electrolytes which do not cross the membrane generally fail to protect. They leave unexplained the need for fast warming, a general requirement of polymer cryopreservation, and none explains why polymers at best generally do not protect more than about 75% of the cells from injury, with the possible exception of erythrocytes. With penetrating cryoprotectants, survival routinely exceeds 90%. There continues to be a need, therefore, for cryopreservation compositions and methods which utilize extracellular cryoprotectants that allow for a range of freezing and thawing procedures.
The present invention provides a general mechanism and method of cryopreservation which utilize extracellular non-penetrating polymer solutions to increase the storage survival of cells in suspension. Compositions useful in the disclosed method are also provided.